Cellulose is one type of hydrocarbon that is the most abundant on the earth. Cellulose is expected to be used as a raw material for producing bioethanol. However, cellulose is macromolecular. Therefore, before cellulose is used as a raw material for producing bioethanol, saccharification is required to turn cellulose into low-molecular-weight saccharides.
As for cellulose saccharification, an acid hydrolysis method, a subcritical water method, and an enzymatic method are known. Among the above methods, the acid hydrolysis method has problems that a reaction tank is damaged by the use of acids, and that the product obtained after the reaction needs to be neutralized, and that saccharides need to be separated from salts after the neutralization. The subcritical water method has problems that a reaction tanks is damaged by subcritical water, and that the reaction proceeds beyond saccharification until cellulose is decomposed into carbon dioxide and water.
Compared with the acid hydrolysis method and the subcritical water method, the enzymatic method has advantages that the impact on a reaction tank is small, and that low-molecular-weight saccharides are obtained as main products, and that the products can be easily separated. The enzymatic method uses cellulase for cellulose saccharification.
Cellulase is a general term for enzymes that catalyze the hydrolysis of cellulose. The known representative examples of cellulase include the following enzymes: cellulase (EC 3.2.1.4), cellulose 1,4-β-cellobiosidase (EC 3.2.1.91), and β-glucosidase (EC 3.2.1.21). Among the above enzymes, cellulase is also referred to as endo-cellulase; cellulose 1,4-β-cellobiosidase is also referred to as cellobiohydrolase.
Some of invertebrates such as termites use cellulase, which is synthesized in their bodies, to assimilate wood. However, when only a single cellulase is used, it is difficult to extract nutrients from the wood. Therefore, scavengers such as termites make use of three types of cellulase (See Non-Patent Document 1 below, the contents of which are incorporated herein by reference). That is, those three types are: endo-cellulase that catalyzes the hydrolysis of cellulose molecules into cellooligosaccharides; cellobiohydrolase that catalyzes the hydrolysis of cellooligosaccharides into cellobiose; and β-glucosidase that catalyzes the hydrolysis of cellobiose into glucose.
The use of a mixture of the three types of cellulase, i.e. endo-cellulase, cellobiohydrolase, and β-glucosidase, makes it theoretically possible to consistently produce glucose from crystalline cellulose such as wood. However, such glucose consistent production has yet to be put into practical use.
In general, if a mixture of two or more purified enzymes is employed, the following conditions need to apply: Condition 1 in which the reaction products of each enzyme do not inhibit the reaction of the other enzymes; and Condition 2 in which the optimum temperatures and optimum pHs of each enzyme need to be consistent. However, endo-cellulase is inhibited by cellobiose and glucose, which are reaction products of cellobiohydrolase and β-glucosidase. Moreover, cellobiohydrolase is inhibited by glucose, which is a reaction product of β-glucosidase. Accordingly, if the mixture of the three types of cellulase, i.e. endo-cellulase, cellobiohydrolase, and β-glucosidase, is used for cellulose saccharification, the above Condition 1 cannot be satisfied (See Non-Patent Document 2 below, the contents of which are incorporated herein by reference).
In view of the above Condition 1, it is hoped that endo-cellulase produced by alkalophilic bacteria, which is not inhibited by cellobiose, will be used (See Patent Document 1 below, the contents of which are incorporated herein by reference). However, the optimum pH of the endo-cellulase derived from alkalophilic bacteria is close to the alkaline side. Therefore, if the reaction takes place under alkaline conditions in line with such a property of alkaline endo-cellulaseendo-cellulase, cellooligosaccharides, which are reaction products of the endo-cellulaseendo-cellulase, are isomerized. As a result, the substrate of cellobiohydrolase, which is to be subjected to a subsequent reaction, is inappropriate. Moreover, the activity of typical β-glucosidase is low under alkaline conditions. Accordingly, if the endo-cellulase derived from alkalophilic bacteria is use to meet the above Condition 1, then the above Condition 2 will not become satisfied.
There has not yet been a mixture of endo-cellulase, cellobiohydrolase, and β-glucosidase that would satisfy the above Conditions 1 and 2. Accordingly, a process of consistently producing glucose from natural cellulose such as wood by using the three types of cellulase has not yet been realized. Meanwhile, attempts have been made to carry out saccharification of natural cellulose such as wood in a single enzyme system by using cellulase that has activity to hydrolyze non-crystalline cellulose molecules (See Patent Documents 2 and 3 below, the contents of which are incorporated herein by reference).
As a scavenger that can assimilate, like termites, wood by utilizing cellulase, the amphipod Hirondellea gigas is known (See Non-Patent Document 3 below, the contents of which are incorporated herein by reference). Hirondellea gigas is one type of gammaridean amphipods that live in the world's deepest part of the Challenger Deep in the Mariana Trench. The research on Hirondellea gigas became popular after unmanned probe “Kaiko” succeeded in capturing more than 100 individuals of Hirondellea gigas in 1998 (See Non-Patent Document 4 below, the contents of which are incorporated herein by reference).
The crushed products of Hirondellea gigas have so far been known to have the activity of protease, α-glucosidase, lipase, amylase, cellulase, and glucomannan degrading enzyme. However, there are no reports that those enzymes have been individually isolated (See Non-Patent Document 5 below, the contents of which are incorporated herein by reference).